Herpes simplex viruses (HSV) types 1 and 2 encode their own ribonucleotide reductases (RNRs) (EC 1.17.4.1) to convert ribonucleoside diphosphates into the corresponding deoxyribonucleotides. Like other iron-dependent RNRs, the viral enzyme is formed by the reversible association of two distinct homodimeric subunits. The carboxy terminus of the RNR small subunit (R2) is critical for subunit association and synthetic peptides containing these amino-acid sequences selectively inhibit the viral enzyme by preventing subunit association. Increasing evidence indicates that the HSV RNR is important for virulence and reactivation from latency. Previously, we reported on the design of HSV RNR inhibitors with enhanced inhibitory potency in vitro. We now report on BILD 1263, which to our knowledge is the first HSV RNR subunit-association inhibitor with antiviral activity in vivo. This compound suppresses the replication of HSV-1, HSV-2 and acyclovir-resistant HSV strains in cell culture, and also strongly potentiates the antiviral activity of acyclovir. Most importantly, its anti-herpetic activity is shown in a murine ocular model of HSV-1-induced keratitis, providing an example of potent nonsubstrate-based antiviral agents that prevent protein-protein interactions. The unique antiviral properties of BILD 1263 may lead to the design of new strategies to treat herpesvirus infections in humans.

We have been investigating the potential of a new class of antiviral compounds. These peptidomimetic derivatives prevent association of the two subunits of herpes simplex virus (HSV) ribonucleotide reductase (RR), an enzyme necessary for efficient replication of viral DNA. The compounds disclosed in this paper build on our previously published work. Structure-activity studies reveal beneficial modifications that result in improved antiviral potency in cell culture in a murine ocular model of HSV-induced keratitis. These modifications include a stereochemically defined (2,6-dimethylcyclohexyl)amino N-terminus, two ketomethylene amide bond isosteres, and a (1-ethylneopentyl)amino C-terminus. These three modifications led to the preparation of BILD 1351, our most potent antiherpetic agent containing a ureido N-terminus. Incorporation of the C-terminal modification into our inhibitor series based on a (phenylpropionyl)valine N-terminus provided BILD 1357, a significantly more potent antiviral compound than our previously published best compound, BILD 1263.

The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (nu/nu) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1) dlsptk and PAAr5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1 dlsptk and PAAr5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAAr5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1 dlsptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1 dlsptk and PAAr5 infections. The effect of BILD 1633 SE against HSV-1 PAAr5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 106 and 107 PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1dlsptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1 dlsptk and PAAr5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.

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